Re-endothelialization of rat lung scaffolds through passive, gravity-driven seeding of segment-specific pulmonary endothelial cells.

Effective re-endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re-endothelialized scaffolds that haemorrhage and become thrombogenic upon implantation. Herein, gravity-driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells were seeded into the pulmonary artery by either gravity-driven, variable flow perfusion seeding or pump-driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re-lining of the vascular walls while perfusion pump-driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells and rat pulmonary vein endothelial cells attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells deposited mostly in microvessels. Combination seeding of these cells led to positive vascular endothelial cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re-endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary-alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. Copyright © 2016 John Wiley & Sons, Ltd.

A nonhuman primate model of lung regeneration: detergent-mediated decellularization and initial in vitro recellularization with mesenchymal stem cells.

Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (∼92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.